Ignacio Díaz Martínez

Drug target validation of the trypanothione pathway enzymes through metabolic modelling

A kinetic model of trypanothione [T(SH)2] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near‐physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)2 synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)2‐dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)2 synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ‐glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58–0.7 and 0.49–0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)2‐dependent antioxidant machinery were determined. The concentration of reduced T(SH)2 was controlled by TryR (0.98) and oxidative stress (−0.99); however, γECS and TryS also exerted control on the cellular level of T(SH2) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)2 synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi‐target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites.

Performance levels of four Latin American laboratories for the serodiagnosis of Chagas disease in Mexican sera samples.

In nearly all of the previous multicentre studies evaluating serological tests for Trypanosoma cruzi infection, sera samples from Central or South American countries have been used preferentially. In this work we compared the reliability of the serological tests using Mexican sera samples that were evaluated in four independent laboratories. This included a reference laboratory in Brazil and three participant laboratories, including one in Central America and two in Mexico. The kappa index between Brazilian and Honduran laboratories reached 1.0 and the index for the Mexican laboratories reached 0.94. Another finding of this study was that the source of antigen did not affect the performance of the serological tests.

Microsatellite and mini-exon analysis of Mexican human DTU I Trypanosoma cruzi strains and their susceptibility to nifurtimox and benznidazole.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and it affects as many as 10 million people in North and South America, where it represents a major public health problem. T. cruzi is a parasite with high genetic diversity, and it has been grouped into 6 discrete typing units (DTUs), designated as T. cruzi I (TcI) to T. cruzi VI (TcVI). Mexican isolates from humans and from vector insects have been primarily found to be TcI, and these isolates are likely to be the strains that cause the clinical manifestations observed in Mexico. However, genetic characterization and drug susceptibility assays are limited in Mexican TcI strains. In this work, 24 Mexican T. cruzi strains, obtained primarily from humans, were studied with 7 locus microsatellites and mini-exon gene by PCR. Also, drug susceptibility was evaluated by growth and mobility assays. All of the human strains belonged to TcI, and they could be further grouped through microsatellite analysis into 2 subgroups (microsatellite genotypes 1 and 2), which were not related to the host clinical status or biological origin of the strain. Two strains, both from wild mammals, belonged to the TcII-TcVI groups; these strains and the CL Brener strain constituted microsatellite genotype 3. The number of alleles in each locus was lower than reported for South American strains, and a departure from the Hardy-Weinberg equilibrium was observed. The susceptibility of these strains to nifurtimox and benznidazole was heterogeneous. T. cruzi strains characterized as microsatellite genotypes 2 and 3 were significantly more susceptible to benznidazole than strains of microsatellite genotype 1. Only 1 Mexican strain resistant to both drugs was found in this study.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

A Novel Crown Ether. 2C60 Complex

The synthesis and characterisation of a novel complex formed by crown ether 2 and 2 molecules of fullerene C60 is reported.

Integral Approach to Evaluation of the Pathogenic Activity of Trypanosoma Cruzi Clones as Exemplified by the Mexican Strain

Comparative histopathological study and analysis of parasite load in different muscle groups were carried out in BALB/c mice during the acute phase of Chagas disease. Activities of C104 clone of T. cruzi strain TPAP/MX/2002/Albarrada and the parental strain were compared. Panoramic 2D-microscopy imaging of sample surface was used and quantitative analysis of parasitism and pathologic damage was performed. The infection rates in various muscle groups were as follows: myocardium = abdominal muscles = lumbar muscles = femoral muscles ← diaphragm for the clone and myocardium⊣abdominal muscles = lumbar muscles = femoral muscles → diaphragm for the parental strain.

Synthesis of 4-methoxy-2-thiomorpholin-4-ylmethyl-1-phenol

Infrared light activation as a non-conventional energy source has become an important method that can be used to carry out a wide range of reactions with short reaction times and high yields.

Complement system contributes to modulate the infectivity of susceptible TcI strains of Trypanosoma cruzi

BACKGROUND Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background.

Synthesis of 1,2,5,7-dithiadiazonan-6-ylidenecyanamide. A New Potential Cysteamine Derivate Radioprotector Compound

Radioprotectors have been described as a chemical compounds that protect certain normal tissues as opposed to tumors.[...]

Synthesis of 4-bromo-2-thiomorpholin-4-ylmethyl-1-phenol

4-bromo-2-thiomorpholin-4-ylmethyl-1-phenol (2) was prepared from 4-bromophenol (1) and thiomorpholine and formaldehyde (2 eq.) and 1 eq. of thiomorpholine.[...]